We are studying the cooperative interactions between cellular and retrovirus-encoded proteins that together regulate RNA synthesis, pre-mRNA processing, and mRNA transport. These interactions are essential for virus replication and for the alterations of intracellular functions that underlie viral pathogenesis. Our research is primarily directed towards identifying the gene products and mechanisms essential for human T-cell leukemia virus type I (HTLV-I) infectivity and oncogenic transformation. We have developed an infectious molecular clone of HTLV-I and have established cell culture systems to monitor virus infectivity, replication and T-cell transformation. We have recently devoloped a single-round infection assay for HTLV-I and are currently examining determinants of virus entry and replication in cell culture. The mechanisms and gene products involved in HTLV-I replication are poorly characterized due to the poor infectivity and inefficient replication of the virus in vitro. In order to define the HTLV-I replication process we have begun to apply quantitative, real-time PCR methods to study replication products in infected cells. We are also examining HTLV-I virion assembly and maturation events to determine their roles in the infectivity process. Primary human T-cells have been infected and immortalized with cloned HTLV-I and with lentivirus vectors that encode HTLV-I regulatory genes in order to examine how cellular signaling networks are reprogrammed by virus gene products. We have begun to examine HTLV-I induced alterations in cellular gene expression profiles in primary human T-cells early after infection. In this regard, we are also investigating HTLV-I Tax-mediated activation of cellular genes controlled by serum response factor (SRF). We found that transcriptional activation mediated by SRF requires the participation of the cellular cofactors Sap1a and CBP. We are investigating how Tax alters the composition and assembly of these enhancer complexes in vitro. We are also asking how Tax modification of the SRF pathway affects the transformation process. We are also comparing the functions and molecular mechanisms of the Rev proteins encoded by various lentiviruses, including human immunodeficiency virus (HIV-1) and its relatives. This research is focused on a comparative analysis of the components and pathways that regulate retroviral mRNA splicing and transport. These processes are mediated by interactions among the viral Rev proteins, cellular factors, and cis-acting RNA elements. A comparison of HIV-1 and equine infectious anemia virus (EIAV) Rev proteins revealed that the latter mediates alternative splicing and nuclear export of viral pre-mRNA. The distantly related Rev proteins share many features in common but also display distinct differences in the ways in which they interact with RNA. We have characterized the primary RNA element bound by EIAV Rev and showed that it is quite different compared to that of other retroviruses. We have also exploited the EIAV system to identify cellular splicing factors that cooperate with Rev in its post-transcriptional regulation of virus gene expression. We have developed an in vitro RNA splicing system to examine the mechanism of Rev-induced alternative splicing. (Project transferred to BRL)AIDS TITLE: Structure and Function of Lentivirus Tat and Rev Proteins.